Column-packing material for gel-permation chromatography, method for its preparation, and applications

ABSTRACT

A column packing material to be used in gel permeation chromatography (GPC) consists of support particles with a mean particle diameter of 1 to 50μ of which the matrix is formed by an inorganic or organic, polymeric and porous hard material with a mean pore diameter of 6 to 50 nm and a pore volume of 0.1 to 3 ml/g, the matrix pores containing chemically bound polymer chains. The column packing material of the invention is characterized in that the polymer chains consist of linear, permeable and not-crosslinked polymers with an upper exclusion limit between 200 and 200,000, the pores of the matrix being entirely filled by the polymer chains. The method of the invention is characterized in that the pores of the support particles are filled by polymer chains chemically bound to the pore surfaces, these polymer chains being permeable to solvents and partly to dissolved substances.

This is a division of application Ser. No. 07/806,216, filed Dec. 13,1991, now U.S. Pat. No. 5,164,427, which is a division of applicationSer. No. 07/596,285, filed Oct. 12, 1990.

The object of the present invention are porous particles filled withpolymeric gels, their manufacturing method and their application aschromatographic supports. By introducing the polymer gel, which forchromatographic purposes is highly advantageous, into a hard, porousmatrix, such materials are exceedingly pressure resistant, non-swellingand evince good storage life even when dry while the chromatographicefficiency is excellent.

Column-packing materials are required gel-permeation chromatography(GPC) that evince high pore density, and of which the pore diameter isspecified and can be controlled accurately. These conditions are met byinorganic, porous supports such as macro-porous silica gels, porousglasses or by macro-porous polymers such as copolymers of styrene anddivinylbenzene. Both types evince large pores (diameters of 60 to 3,000Å) and thereby allow separating substances of higher molecular weights.To separate substances of lower molecular weights a support with smallerpores is required. Such small pores cannot be achieved with theaforementioned supports. They require homogeneously crosslinked polymergels as supports. In that case the substances are separated bydifferential diffusion in the grain wherein the polymer chains build upa mesh lattice and prevent larger substances from diffusing. The basicdrawback of these materials is in their low mechanical strength. Theparticles are quite soft and will deform at higher pressures so that, atsuch higher pressures, the chromatographic column will clog. Because ofthese drawbacks, the intrinsically highly selective and homogeneouslycrosslinked gels have almost disappeared from the market.

Presently special supports are used in this range of molecular weights,but their accurate manufacturing procedure is not disclosed in theliterature. However these supports lack inorganic components. Theirseparation efficiency is very good, their pressure resistance isaverage, their handling properties and storage lives on the other handare poor, and they are among the most expensive materials in HPLC andGPC. They are commercially available as PL-Gel, Styragel, TSK Gel andShodex GPC columns.

Attempts to prepare hard particles with small pores are generally knownbut they have not become commercial products. Pores of silica gel havebeen lined with polymer layers to make the pores smaller (B. Monrabal,Chromatog. Sci. 19, 79, 1981). Diffusion inside these layers was neitherrequired nor intended and therefore mostly highly crosslinked, porouspolymer layers have been used.

Again the literature discloses attempts to deactivate by polymer-coatingthe pore surfaces of porous solids to make them useful in GPC (forinstance B. Sebille et al, European patent application 225829 A2 of 16Jun. 1987, A. Iranov et al, Mol. Genet. Mikrobiol. Viruso, 11, 39, 1987,Chemical Abstracts 108, 34 286 198; Y Komiya et al, Kokai Tokkyo Koho,Japanese patent 55/5941, 17 Jan. 1980). Many other works are known onpreparing column materials evincing chemically bound polymer layers atthe surface of the silica gel and which are meant to be used for ionexchange, reversed phase chromatography, for racemate separation etc.(for instance H Okamura et al,German Offenlegungsschrift 2730771, 12Jan. 1978; I Shinichi et al, U.S. Pat. No. 4,140,653, 20 Feb. 1979;Figge et al, J. Chromatog. 351, 393, 1986; G Wulff et al, ReactivePolymers, 3, 261, 1985; Daicel Chemical Industries Ltd. Kogai TokkyoKoho, Jap. patent kokai 57/147,434, 11 Sep. 1982. Y Kosaka et al,Japanese patent document kokai 51/74694, 28 Jun. 1976; A V Il'ina et al,Russian patent 687081, 25 Sep. 1979).

In all such work, the separation in the chromatographic column is byprinciples other than GPC. Moreover, in all instances, the polymerlayers on the pore surfaces are more or less thin in order that, as isexplicitly explained, the diffusion shall become as good as possible inthe free spaces in the grain.

Now we discovered in surprising manner that porous materials whollyfilled with homogeneous polymer gels make excellent chromatographicsupports. The polymer lattice in the pore polymer gel can be determinedby the number of covalently bound groups containing in turn vinyl groupsand adhering to the pore surfaces of the solid as well as by the lengthof the polymer chains. The manufacturing methods of these particles alsoare an object of this invention.

In the preparation method of the invention, the molecules withpolymerizing double bonds or also the molecules with other functionalgroups reacting into polymerization, polycondensation or polyadditionare chemically bound in concentrations of 0.01 to 6 μmole/cm² to thesurface of porous solids.

Suitable porous solids are materials with an inner volume of 0.1-3 ml/g(preferably between 0.6 and 1.2 ml/g) and an average pore diameter of6-500 nm (preferably 10-50 nm). Between 1 and 50μ, preferably between 3and 10μ, the average particle size preferably shall evince a standarddeviation less than 50%.

Applicable porous materials for instance are silica gel, aluminum oxide,Celite, zeolites, porous glass and furthermore polymers based on vinyl,vinylidene, methacrylate, acrylate or the like, monomers with suitablecrosslinking agents such as divinylbenzene, glycol-dimethylacrylate,methylene-bis-acrylamide or the like. Basically however all porousmaterials with sufficient inertia and mechanical strength may be used.

The bonding of the polymerizing groups also can be carried out byprocedures known from the literature. Among the advantageous bonds arethose halogen- or alkoxy-silanes on silica gel for instance with3-(trimethyoxysilyl)propylmethacrylate or4-(dimethylchlorosilyl)-styrene or of methacryl anhydride on polyhydroxyethylene methacrylate.

The suitable concentration of the polymerizing groups on the poresurfaces is of decisive significance for the application of theinvention, and this shall be discussed in further detail below. Toregulate this concentration, either a varyingly large quantity is usedin the reaction, or the reagent is diluted with a reagent of similarstructure but lacking any polymerizing group. In every case as regardssilica gel, glass, zeolites or Celite, unconverted silanol groups mustbe deactivated subsequently by known methods, for instance usinghexamethyldisilazane or similar reagents. Groups interfering with theultimate application must be deactivated at the surface also for othersolids.

The monomer or the mixture of monomers to be polymerized will be addedin a concentration of 0.1-10 g per g of porous solid so modified.Preferably the monomer volume shall be 5-100% by volume larger than theinside volume of the solid. Next the well volatile solvent is removed ina vacuum of 0.1 to 300 mbars at a temperature of 0°-100° C. In order toapply lesser quantities of monomer, the monomer is additionally dilutedwith a less volatile and inert solvent such as toluene, xylene. Suitablemonomers are all those proper for radial polymerization (olefins,styrene derivatives, acrylic derivatives, methacrylate derivativesetc.). In this case a suitable initiator (peroxides or azo compoundsfrom the literature) is added to the monomer mixture. Then the mixtureis heated for 1-100 h at 30°-120° C. To initiate the polymerization,other methods from the literature also may be employed (anionic,cationic, coordinated polymerizations). However the monomers also may besuch as to make possible polycondensation or polyaddition to build upthe polymer.

After polymerization, the chemically non-bound polymer is exhaustivelyrinsed with a suitable solvent, ie a solvent in which the polymer iswell soluble, and thereupon the porous material filled with the polymeris dried.

Substances prepared in this manner are excellent packing materials forchromatographic columns, in particular for gel permeationchromatography. One of the features of the invention relating to theseGPC materials is that the mesh-lattice of the polymer determining theexclusion limits of the substances to be separated is not controlled byadding conventional bifunctional crosslinking agents. In this case thecrosslinking is of a novel sort by means of the number of adhesiongroups at the surface. As shown by FIG. 1, the exclusion limits of theGPC can be controlled by the number of adhesion groups at the poresurfaces. They may also be controlled conventionally by addingcrosslinking agents, however the chromatographic mass transfer thenshall be significantly less advantageous. Furthermore the exclusionlimits can be controlled to some extent by the number of polymer chainsper cavity or by their degree of polymerization. A lesser number ofpolymer chains is achieved by shortening the polymerization time (FIG.2) or by diluting the monomer mixture with a less volatile inert solventduring deposition on the porous material and during the ensuingpolymerization (FIG. 3). The degree of polymerization of the chains canbe controlled by means of the proportion of initiator, by addingcarriers during polymerization or by the polymerization temperature.Again the exclusion limits are affected thereby (FIG. 4). The masstransfer is more advantageous as the degree of polymerization of thechains is less and the material is more easily packed into the columns.

Further advantages of the invention from the materials so prepared arethe mechanical strength because the intrinsically soft gel phases areprotected by the hard porous structures of the solids. Therefore theymay be used at all ordinary pressures occurring in HPLC. As shown byFIGS. 5 a-c, the separation efficiency at high flows and high pressuresis very good, being a maximum at 1 ml/min, so that separations terminatein 2.4 min. However even at 3 ml/min and 388 bars, separation still isgood and ends after 0.8 min. The particles are easily filled while ofhigh quality into the columns and the column contents practically willnot swell. Solvents may be directly changed during chromatographywithout thereby incurring losses in quality. The packed column even maybe open for some time and be heated to 80° C. and then be reused. Theseparation efficiency does not change. Even sudden jumps in pressurecause no degradation of the column packing.

An especially advantageous porous material is silica gel which can bemade commercially with tight controls as regards internal volume, poredistribution, particle shape and particle-size distribution.

The preparation of the polymeric gel packings reinforced by silica geltakes place in two steps of the method of the invention.

The first step is carried out at a present-day technical level accordingto known procedures, however under new experimental conditions. In thisstep a desired concentration of polymerizing groups shall be bound tothe silica-gel surface. Therefore dry silica gel is brought into contactwith a compound comprising polymerizing groups, either directly ordissolved in a solvent, and by chemical reaction is bound to the silicagel surface. Suitable modifying reagents illustratively are3-(trimethoxysilyl)-propylmethacrylate, 4-(dimethylchlorosilyl)styreneor 4-(trichlorosilyl)-styrene. 4-(dimethylchlorosilyl)styrene isespecially well suited. The concentration of these groups furthermorecan be varied by adding varying amounts of trimethylchloro-silane. Afterthe modified silica gel has been thoroughly washed, the residual, stillaccessible silanol groups on the silica gel surface are end-capped inknown manner using a suitable reagent. Suitable reagents for ating thesilanol groups are illustratively trimethylchlorosilane,1,1,1,3,3,3-hexamethylenedisilazane (HMDS) or ethoxytrimethylsilane.HMDS is especially gentle.

In the second step of the method of the invention, suitable monomers inconcentrations from 0.1 to 1,000% by weight related to the originalsilica gel are added to the silica gel modified in the manner of theinvention to produce polymerization. The monomer already contains asuitable initiator in a concentration of 0.01 to 10% by weight relativeto the monomer. Once the monomers have been uniformly spread on thesilica-gel surface, polymerization begins. In practice polymerization isachieved using AIBN as initiator at temperatures between 55° and 70° C.Following polymerization, the chemically unbound, eluting polymer isrinsed-out using a suitable solvent. Methylene chloride, toluene ortetrahydrofuran are especially applicable.

The Examples below of practically applications elucidate, withoutimplying restriction, the method of the invention.

EXAMPLE 1

Activation of the silica gel used, namely Matrex Silica Si (mean porediameter=25 nm, particle size 0.01 mm) made by Amicon Co. (Witten, W.Germany) is carried out by boiling for 2 h in 5% nitric acid, by washingto neutrality with distilled water and by drying at 120° C. in thevacuum oven.

2.5 g of the silica gel so pre-treated were suspended in 25 ml of drycarbon tetrachloride and following addition of 0.021 ml of dry pyridineand 0.023 ml of 4-(dimethylchlorosilyl)-styrene, the mixture was shakenfor 2 h at room temperature. After filtering and washing with drymethylene chloride, drying of the modified silica gel was carried out at40° C. in the vacuum oven. The carbon content of the silica gel somodified was 0.9%, that is, the concentration of the anchor group wasabout 9% relative to the maximum possible surface coating with anchorgroups.

2.5 ml of 1,1,1,3,3,3-hexamethylenedisilazane (HMDS) were added to asuspension of the modified silica gel in 25 ml of dry carbontetrachloride and the mixture was shaken for 2 h at room temperature.Following filtration and washing with methylene chloride, drying wascarried out at 40° C. in the vacuum oven. The carbon content of thesilica gel so modified and so end-capped was 4.08%.

3.977 g of styrene and 0.209 g of azo-bis-isobutyronitrile (AIBN) wereadded to a suspension of 2.2 g of the modified and end-capped silica gelin 25 ml of dry methylene chloride. Following shaking of the suspensionand removal of the methylene chloride in a rotary evaporator atwater-jet vacuum, further repeated purging with N₂ the reaction flaskand ensuing application of a vacuum of about 120 mbars, polymerizationwas carried out for three days at 70° C.

After polymerization the silica gel polymer was suspended in 50 ml ofmethylene chloride and shaken for 6 h. After filtration and thoroughwashing, drying was carried out at 40° C. in the vacuum oven. The carboncontent of the silica gel was 24.5%, that is, in relation to the initialsilica gel, a total of 37.8% of organic compounds and polymer had beenadded.

EXAMPLE 2

The activation of the silica gel used, i.e. Matrex Silica SI (mean porediameter 25 nm, particle size 0.01 mm) from Amicon Co. (Witten, WestGermany) was carried out by boiling for 2 h in 5% nitric acid, washingto neutrality with distilled water and drying in the vacuum oven at 120°C.

Next, 7.5 g of this pre-treated silica gel were suspended in 75 ml ofdry carbon tetrachloride, then 1.95 ml of dry pyridine and a mixtureconsisting of 0.261 ml of 4-(dimethylsilyl)-styrene and 1.910 ml ofchloro-trimethylsilane were added and the substance was allowed to standfor 20 h at room temperature with occasional shaking. Followingfiltration and washing with dry methylene chloride, the modified silicagel was dried at 40° C. in the vacuum oven. The carbon content of thesilica gel so modified was 4.77%.

16.703 g of styrene and 0.879 g of azo-bis-isobutyronitrile (AIBN) wereadded to a suspension consisting of 7.0 g of the modified silica gel in70 ml of dry methylene chloride. After shaking the suspension andremoving the methylene chloride using the rotary evaporator at water jetvacuum, also by repeatedly purging with N₂ the reaction flask andsubsequent application of a vacuum of about 120 mbars, polymerizationwas carried out at 70° C. for three days.

After the polymerization, the silica gel was suspended twice in 100 mlof methylene chloride and shaken for 6 h. Following filtration andthorough washing, drying was carried out at 40° C. in the vacuum oven.

The carbon content of the silica-gel polymer was 31.9%. In relation tothe initial silica gel, a total of 55.6% of organic compounds andpolymer had been added.

EXAMPLE 3

The silica gel used, namely Matrex Silica Si (mean pore diameter 25 nm,mean particle size 0.01 mm) made by Amicon Co. (Witten, West Germany)was carried out by boiling for 2 h in 5% nitric acid, by washing toneutrality with distilled water and drying in the vacuum oven at 120° C.

5 g of the silica gel so pre-treated were suspended in 50 ml of drycarbon tetrachloride and following addition of 0.273 ml of3-(trimethoxysilyl)-propylmethacrylate, the mixture was heated toboiling with reflux for 8 h. Following filtration and washing with drymethylene chloride, the modified silica gel was dried at 40° C. in thevacuum oven. The carbon content of the silica gel so modified was 5.31%.

5 ml of 1,1,1,3,3,3-hexamethylene disilazane (HMDS) were added to asuspension of the modified silica gel in 50 ml of dry carbontetrachloride and this mixture was agitated for 2 h at room temperature.Following filtration and washing with methylene chloride, drying wascarried out at 40° C. in the vacuum oven. The carbon content of thesilica gel so modified and end-capped was 5.64%.

3.268 g of 2-hydroxyethyl methacrylate and 0.084 g ofazo-bis-isobutyronitrile (AIBN) were added to a suspension of 4 g of themodified and end-capped silica gel in 50 ml of dry methylene chloride.After shaking the suspension and removing the methylene chloride usingthe rotary evaporator at water-jet vacuum, also upon repeatedly purgingthe reaction flask and then applying a vacuum of about 120 mbars,polymerization was carried out at 70° C. for three days. After thispolymerization the silica- gel polymer was suspended in 100 ml ofmethanol and agitated for 6 h. Following filtration and thorough washingof the material, drying was carried out at 40° C. in the vacuum oven.The carbon content of the silica-gel polymer was 27.67%. Computed inrelation to the initial silica gel, approximately 99.2% of organiccompounds and polymer had been added.

EXAMPLE 4

The silica gel used, namely Nucleosil 300-5 (mean pore diameter 30 nm,particle size 0.005 mm) made by Macherey und Nagel (Duren, WestGermany), was activated by boiling for 2 h in 5% nitric acid, by washingto neutrality with distilled water and drying in the vacuum oven at 120°C.

5.0 g of the silica gel so pre-treated were suspended in 50 ml of drycarbon tetrachloride, and, following addition of 0.7 ml of dry pyridineand a mixture of 0.070 ml of 4-(dimethylsilyl)styrene and 0.466 ml ofchlorotrimethylsilane, this material was allowed to stand at roomtemperature for 20 h with occasional agitation. After filtration andwashing with dry methylene chloride, the modified silica gel was driedat 40° C. in a vacuum oven. The carbon content of the silica gel somodified was 1.59%.

9.09 g of styrene and 0.478 g of azo-bis-isobutyronitrile (AIBN) wereadded to a suspension consisting of 5.0 g of the modified silica gel in50 ml of dry methylene chloride. After shaking the suspension andremoving the methylene chloride by means of the rotary evaporator atwater-jet vacuum, also upon purging with N₂ the reaction flask severaltimes and then applying a vacuum of about 120 mbars, polymerization wascarried out for three days at 70° C.

Upon polymerization, the silica-gel polymer was suspended twice in 100ml of methylene chloride and agitated for 6 h. After filtration andthorough washing, drying was carried out at 40° C. in the vacuum oven.

The carbon content of the silica gel polymer was 16.81%. Relative to theinitial silica gel, a total of 22.3% of organic compounds and polymerwere computed to have been added.

EXAMPLE 5

The silica gel used, namely Matrex Silica Si (mean pore diameter 25 nm,particle size 0.01 mm) made by Amicon Co. (Witten, West Germany), wasactivated by boiling for 2 h in 5% nitric acid, washing to neutralitywith distilled water and drying in the vacuum oven at 120° C.

5 g of the silica gel so pre-treated were suspended in 50 ml of drycarbon tetrachloride and, following addition of 1.95 ml of dry pyridineand 2.13 ml of 4-(dimethylchlorosilyl)-styrene, this mixture wasagitated for 2 h at room temperature. After filtration and washing withdry methylene chloride, drying of the modified silica gel was carriedout at 40° C. in the vacuum oven. The carbon content of the silica gelso modified was 9.79%.

5.0 ml of 1,1,1,3,3-hexamethylenedisilazane (HMDS) were added to asuspension of the modified silica gel in 50 ml of dry carbontetrachloride and this mixture then was shaken for 2 h at roomtemperature. Following filtration and washing with methylene chloride,drying was carried out at 40° C. in the vacuum oven. The carbon contentof the silica gel so modified and end-capped was 10.0%.

5.966 g of styrene and 0.314 g of azo-bis-isobutyronitrile (AIBN) areadded to a suspension composed of 5 g of the modified and end-cappedsilica gel in 50 ml of dry methylene chloride. After agitating thesuspension and removing the methylene chloride at the rotary evaporatorin water-jet vacuum, and after repeatedly purging with N₂ the reactionflask and then applying a vacuum of about 120 mbars, polymerization wascarried out for three days at 70° C.

Following the polymerization, the silica-gel polymer was suspended in 75ml of methylene chloride and shaken for 6 h. After filtration andthorough washing, drying took place at 40° C. in the vacuum oven. Thecarbon content of the silica-gel polymer was 46.71%, that is, withrespect to the initial silica gel, a total of 102.6% of organiccompounds and polymer had been added.

EXAMPLE 6

Activation of the silica gel used, namely LiChrosorb Si 100 (mean porediameter 10 nm, particle size 0.01 mm) made by Merck Co. (Darmstadt,West Germany) was carried out by boiling for 2 h in 5% nitric acid,washing to neutrality and drying in the vacuum oven at 120° C.

5.0 g of the silica gel so pre-treated were suspended in 50 ml of drycarbon tetrachloride and following addition of 1.94 ml of dry pyridineand 2.279 ml of 4-(dimethylchlorosilyl)-styrene, this mixture was shakenfor 2 h at room temperature. After filtration and washing with drymethylene chloride, drying of the modified silica gel was carried out at40° C. in the vacuum oven. The carbon content of the silica gel somodified was 9.47%.

5.0 ml of 1,1,1,3,3,3-hexamethylenedisilazane (HMDS) were added to asuspension of the modified silica gel in 50 ml of dry carbontetrachloride and this mixture was then agitated for 2 h at roomtemperature. After filtration and washing with methylene chloride,drying was carried out at 40° C. in the vacuum oven. The carbon contentof the silica gel so modified and end-capped was 9.85%.

11.931 g of styrene and 0.62 g of azo-bis-isobutyronitrile (AIBN) wereadded to a suspension composed of 5 g of the modified and end-cappedsilica gel in 50 ml of dry methylene chloride. After agitating thesuspension and removing the methylene chloride using the rotaryevaporator and at water-jet vacuum, and after repeatedly purging with N₂the reaction flask and then applying a vacuum of about 120 mbars,polymerization was carried out for three days at 70° C.

Following the polymerization, the silica-gel polymer was suspended in 75ml of methylene chloride and shaken for 6 h. After filtration andthorough washing, drying was carried out in the vacuum oven at 40° C.The carbon content of the silica-gel polymer was 46.58% which relativeto the original silica gel is computed to amount to a total addition of102% of organic compounds and polymer.

EXAMPLE 7

The silica gel used, namely Martex silica Si (mean pore diameter 25 nm,particle size 0.01 mm) made by Amicon Co. (Witten, W. Germany), wasactivated by boiling for 2 h in 5% nitric acid, by washing to neutralitywith distilled water and by drying at 120° C. in the vacuum oven.

5 g of the pre-treated silica gel were suspended in 25 ml of dry carbontetrachloride and, following addition of 1.95 ml of dry pyridine and of2.164 ml of 4-(dimethylchlorosilyl)-styrene, the mixture was agitatedfor 2 h at room temperature. After filtration and washing with drymethylene chloride, the modified silica gel was dried at 40° C. in thevacuum oven. The carbon content of the silica gel so modified was 9.79%.

5 ml of 1,1,1,3,3,3-hexamethylenedisilazane (HMDS) were added to asuspension of the modified silica gel in 50 ml of dry carbontetrachloride and the mixture was agitated for 2 h at room temperature.Following filtration and washing with methylene chloride, drying wascarried out at 40° C. in the vacuum oven. The carbon content of thesilica gel so modified and end-capped was 10.0%.

3.18 g of styrene, 9.104 g of toluene and 0.167 g ofazo-bis-isobutyronitrile (AIBN) were added to a suspension composed of 4g of the modified and end-capped silica gel in 40 ml of dry methylenechloride. After shaking the suspension and removing the methylenechloride by means of the rotary evaporator and at water-jet vacuum, andafter repeatedly purging the reaction flask with N₂ and ensuingapplication of a vacuum of about 120 mbars, the polymerization wascarried out at 70° C. for three days.

Following the polymerization, the silica gel polymer was suspended in 50ml of methylene chloride and agitated for 6 h. After filtration aridthorough washing, drying was carried out at 40° C. in the vacuum oven.The carbon content of the silica-gel polymer was 31.3%, that is, inrelation to the initial silica gel, a total of 51.5% of organic compoundand polymer were computed to have been added.

EXAMPLE 8

The hydroxyethylmethacrylate of mean pore diameter of 100 nm (HEMA fromPolymer Standards, Mainz, West Germany) was dried at 40° C. in thevacuum oven.

5 g of the dried HEMA 1000 were suspended in 100 ml of dry methylenechloride and following addition of 0.1 ml of dry pyridine and 0.05 ml ofmethacrylic-acid chloride, the mixture was agitated at room temperaturefor 2 h. After filtration and washing with methylene chloride, dryingwas carried out at 40° C. in the vacuum oven.

5.0 g of 2-hydroxyethylmethacrylate and 0.263 g of AIBN were added to asuspension of 50 ml of dry methylene chloride and of 5 g of the modifiedHEMA. After shaking the suspension and removing the methylene chlorideusing the rotary evaporator at water-jet vacuum, and after purging thereaction flask several times with N₂, and thereupon applying a vacuum ofabout 120 mbars, polymerization was carried out at 70° C. for threedays.

Following polymerization, the modified HEMA was suspended in 100 ml ofmethanol and agitated for 6 h. After filtration and thorough washing,drying was carried out at 40° C. in the vacuum oven.

EXAMPLE 9

Activation, modification and polymerization of the silica gel Matrexsilica Si (mean pore diameter 25 nm, particle size 0.005 mm) from AmiconCo. (Witten, W. Germany) takes place as in Example 2.

3.5 g of the polymerically modified silica gel of Example 2 aresuspended in 125 ml of dry xylene and following addition of 2.5 g of1,1,1,3,3,3-hexamethyldisilazane (HMDS) the mixture is boiled withreflux for 20 h. After filtering and washing with methylene chloride,drying takes place at 40° C. in the vacuum oven.

    ______________________________________                                        Accurate material data:                                                       ______________________________________                                        Base material:     Matrex    silica Si                                        Mean pore diameter:                                                                              25 nm                                                      Particle size:               0.005 mm                                         Specific surface:  292 m.sup.2 /g                                             Modified silica gel                                                           Specific surface:  215.4 m.sup.2 /g                                           Carbon content:    4.77%                                                      Polymer-modified silica gel                                                   Specific surface:  151.4 m.sup.2 /g                                           Carbon content:    31.9%                                                      Column packing (by the slurry method)                                         2.25 g of polymerically modified silica gel                                   45 ml of methanol/dioxan (2/1)                                                Mobile solvent:    methanol/dioxan (2/1)                                      Pressure:          300 bars                                                   Flow:              about 4.5 ml/min                                           Duration           2.5 h                                                      Column dimensions: 250 × 4 mm                                           ______________________________________                                    

LEGENDS OF FIGURES

FIG. 1: Variation of anchor group concentrations

Comparison of eluted volumes of chromatographic materials for GPC,differing only in the concentrations of the anchor groups(4-vinyl-phenyl-siloxane) and thereby resulting in different exclusionlimits for substances of different molecular weights. The reference isnon-modified silica gel (pure). The fraction of maximum possibleanchor-group concentration (1/3; 1/11; 1/31; 1/100) is stated each time.

FIG. 2: Variation of polymerization time

For a silica gel with 1/11 of the possible anchor groups, thepolymerization times were varied while keeping other conditions constantand the GPC behavior of the resulting materials was ascertained.

pure=reference silica gel, unmodified; durations: 1 h; 2 h; 4 h.

FIG. 3: Variation in inert solvents

Other conditions being kept constant, the monomer was diluted duringmanufacture with an inert solvent (toluene) and the GPC behavior of theresulting materials was ascertained. 1/0=no dilution; in the othertests, dilution to 1, 3, 7, 11 parts by volume of monomer to 15 parts byvolume of inert solvent.

FIG. 4: Variation in AIBN concentration

Keeping the other conditions constant, the initiator (AIBN)concentration was varied and thereupon the GPC behavior of the materialswas ascertained.

There were 0.1, 1.0, 2.5 and 5% by weight of AIBN per monomer portion.

FIGS. 5 a-f

These are gel-chromatographic separations of polystyrene 111 000(PST-111000), polystyrene 5000 (PST 5000), tristearin, triacontane (C30), eicosane (C20), tetradecane (C 14), octane (C 8) and benzene atvarious flows. The column is that of Example 9.

We claim:
 1. Column-packing material for gel-permation chromatography,comprising:a) support particles having a plurality of pores, saidparticles formed of an inorganic or organic, polymeric material andhaving a mean particle diameter of 1 to 50μ, a mean pore diameter of 6to 500 nm, and a pore volume of 0.1 to 3 mg/l; b) at least some of saidpores having been chemically modified for providing bonding sitestherein; and, c) polymer chains chemically bonded to said bonding sites,said polymer chains comprised of a material permeable to solvent and topartially dissolved substances.
 2. The material of claim 1, furthercomprising:a) first anchor groups covalently bonded to the surface ofsaid particles; and, b) a polymerized mixture of monomers and initiatorbonded to said anchor groups and filling the free spaces of said pores.3. The material of claim 1, wherein:a) said support particles having amean pore diameter of 10 to 200 nm, a pore volume of 1.3 to 2 mg/l perliter, and a mean particle size of 3 to 20μ.
 4. The material of claim 1,wherein:a) the number of polymer chains bonded to said particles is fromabout 0.01 to about 10 μmole/m².
 5. The material of claim 4, wherein:a)the number of polymer chains bonded to said particles not exceeding 3.0μmole/m².
 6. The material of claim 1, wherein:a) said support particlescomprised of a material selected from the group consisting of silicagel, aluminum oxide, glass, zeolite, Celite, polymers based on a memberselected from the group consisting of vinyl, vinylidene, methacrylate,and acrylate, and monomers having a cross-linking agent selected fromthe group consisting of divinylbenzene, glycol dimethacrylate, andmethylene-bis-acrylamide.
 7. The material of claim 1, wherein:a) saidsupport particles are formed from silica gel.
 8. The material of claim1, wherein:a) said support particles are formed from a member selectedfrom the group consisting of silica gel, glass, zeolite, and Celite;and, b) unconverted silanol groups of said particles having beendeactivated.
 9. The material of claim 8, wherein:a) said pores havingbeen modified by reaction with a reagent selected from the groupconsisting of 3-(trimethoxysilyl)-propylmethacrylate,4-(dimethylchlorosilyl)styrene, 4-(trichlorosilyl)-styrene, and4-(dimethylchlorosilyl).
 10. The material of claim 9, wherein:a) silanolgroups remaining after modification having been end-capped by reactionwith a reagent selected from the group consisting oftrimethylchlorosilane, 1,1,1,3,3,3-hexamethylenedisilane, andethoxytrimethylsilane.
 11. The material of claim 1, wherein:a) saidpolymer chains are selected from the group consisting of styrene,styrene derivatives, and acrylic monomers selected from the groupconsisting of acrylesters, acrylamides, and methylmethacrylates.
 12. Thematerial of claim 1, wherein:a) said polymer chains comprised of linear,permeable, not cross-linked polymers having an upper exclusion limit ofbetween about 200 to about 200,000.